Advances in the biological, biomedical and pharmaceutical sciences have accelerated the pace of research and diagnostics unparalleled to the past. With whole genome sequences becoming quickly and successively available, the assembly of large libraries of small molecules, and the ability to move pharmaceutical development, clinical diagnostic tests and basic research from a reductionist to a whole system approach demands assays that facilitate high throughput analyses. Molecules no longer need to be singly analyzed for their effects on a lone process; instead, the effects of many molecules on several biological systems can be studied simultaneously—if appropriate, fast, reliable, and accurate assays are available.
Due to their physiological relevance, variety and ubiquitousness, transferases, especially kinases, have become one of the most important and widely studied families of enzymes in biochemical and medical research. Studies have shown that protein and lipid kinases are key regulators of many cell functions, including signal transduction, transcriptional regulation, cell motility, cell division and cellular responses to drugs, toxins, and pathogens.
Protein kinases play crucial roles in the modulation of a wide variety of cellular events. These enzymes act by transferring phosphate residues to certain amino acids in intracellular polypeptides, to bring about the activation of these protein substrates, and set in motion a cascade of activation controlling events including the growth, differentiation and division of cells. Protein kinases have been extensively studied in the field of tumour biology. A lack of controlled activity of kinases in cells is believed to lead to the formation of tumours. The pharmaceutical industry is constantly in search of drugs that target these kinases, to help with the treatment of a wide variety of tumours. There are at least 1200 protein kinases that are involved in the regulation of cell functions. They occur as both transmembrane and cytosolic enzymes and they phosphorylate serine, threonine and tyrosine amino acid residues. Based on these substrate specificities the kinases are divided into two groups, the serine/threonine kinases and tyrosine kinases.
Serine/threonine kinases, includes cyclic AMP and cyclic GMP dependent protein kinases, calcium and phospholipid dependent protein kinase, calcium and calmodulin-dependent protein kinases, casein kinases, cell division cycle protein kinases and others. These kinases are usually cytoplasmic or associated with the particulate fractions of cells, possibly by anchoring proteins.
Tyrosine kinases phosphorylate tyrosine residues. These particular kinases are present in much smaller quantities but play an equally important role in cell regulation. These kinases include several soluble enzymes such as src family of protein kinases, and receptors for growth factors such as epidermal growth factor receptor, insulin receptor, and platelet derived growth factor receptor and others. Studies have indicated that many tyrosine kinases are transmembrane proteins with their receptor domains located on the outside of the cell and their kinase domains on the inside.
Lipid kinases also play important roles in the intracellular signal transduction, and have been grouped into four major classes. Exemplary lipid kinases include PI3 kinases, and phosphatidylinositol 4-kinases.
Current types of assays used to measure kinase activity and to detect potential kinase inhibitors are cumbersome and costly. These assays include Fluorescence Resonance Energy Transfer (FRET) assays, Fluorescent Plarization (FP) assays, and assays based on radioactivity such as Scintillation Proximity Assay (SPA).
FRET assays used to detect kinase activity utilize a protein substrate that has two linked fluorescent molecules. The two molecules are in close proximity, separated by a fixed distance. The energy of an excited electron in one molecule (the donor) is passed to an adjacent molecule (the acceptor) through resonance. The ability of a higher energy donor flourophore to transfer energy directly to a lower energy acceptor molecule causes sensitized fluorescence of the acceptor molecule and simultaneously quenches the donor flouorescence. In this case, the fluorescence of the donor is “quenched” by the proximity to the acceptor and the energy of the donor is transferred to the acceptor in a non-radiative manner. The efficiency of energy transfer is dependent on the distance between the donor and acceptor chromophores according to the Forster equation. In most cases, no FRET is observed at distances greater than 100 angstroms and thus the presence of FRET is a good indicator of close proximity.
In order for FRET to be useful, the fluorescence of the acceptor molecule must be significantly different from the fluorescence of the donor. A useful FRET based protein substrate may include a separation of the two fluorescent molecules via a peptide linker that maintains specificity for an endopeptidase that is capable of cleaving the peptide linker between the two fluorophores. If the peptide is phosphorylated, then the enzyme may not cleave the protein or may cleave it at a reduced rate, keeping the fluorescent molecules in close proximity such that quenching occurs. On the other hand, if the protein is not phosphorylated, then the endopeptidase cleaves the protein substrate, releasing the two fluorescent molecules such that the quenching is alleviated, and the two fluorescent molecules fluoresce independently. The FRET assay requires peptide substrates that must be carefully engineered to meet these requirements. That is, the peptide substrates must contain the enzyme recognition site required for the endopeptidase, the distance between the two fluorophores must be within the range to allow FRET to occur and the fluorescent molecules must be paired in such a way that donor fluorescence is significantly quenched, minimizing background fluorescence from the donor. Furthermore, the fluorescence of the starting material (the “quenched” substrate) must be siginificantly different from the product (the “released” non-quenched product). These requirements make a FRET based assay cumbersome and costly.
FP assays are based on binding of a high affinity binding reagent, such as an antibody, a chelating atom, or the like, to a fluorescent labeled molecule. For example, an antibody that binds to a phosphorylated fluorescent labeled peptide but not a non-phosphorylated fluorescent labeled peptide can be used for a kinase assay. When the fluorescent label is excited with plane polarized light, it emits light in the same polarized plane as long as the fluorescent label remains stationary throughout the excited state (duration of the excited state varies with fluorophore, and is 4 nanoseconds for fluoroscein). However, if the excited fluorescent label rotates or tumbles out of the plane of polarization during the excited state, then light is emitted in a different plane from that of the initial excitation state. If polarized light is used to excite the fluorophore, the emission light intensity can be monitored in both the plane parallel to the plane of polarization (the excitation plane) and in the plane perpendicular to the plane of polarization. The degree to which the emission intensity moves from the parallel to the perpendicular plane is related to the mobility of the fluorescent labeled molecule. If the fluorescent labeled molecules are large, such as when they are bound to the binding reagent, the fluorescent labeled molecules move little during the excited state interval, and emitted light remains highly polarized with respect to the excitation plane. If the fluorescent labeled molecules are small, such as when no binding reagent is bound to the fluorescent labeled molecules, the fluorescent labeled molecules rotate or tumble faster, and the resulting emitted light is depolarized relative to the excitation plane. Thus, an FP assay requires a high affinity binding reagent, e.g., an antibody, capable of binding with high specifity to the fluorescent labeled molecule. The time consuming and costly optimization of antibody binding with specific fluorescent labeled molecules such as peptides is required where antibodies are used. Additionally, the FP assay there is the potential for phosphorylated protein and other reaction components, e.g., lipids and detergents, to interfere with the polarization.
Kinase assays that use radioactive labels include SPA. In SPA, modified ligand-specific or ligand-capturing molecules are coupled to fluoromicrospheres, which are solid-phase support particles or beads impregnated with substances that emit energy when excited by radioactively labeled molecules. When added to a modified ligand such as radio-labeled phosphopeptide in a mixture with nonphosphorylated peptide, only the phosphopeptide is captured on a fluoromicrosphere, bringing any bound radiolabeled peptide close enough to allow the radiation energy emitted to activate the fluoromicrosphere and emit light energy. If the concentration of fluoromicrospheres is optimized, only the signal from the radio-labeled ligand bound to the target is detected, eliminating the need for any separation of bound and free ligand. The level of light energy emitted may be measured in a liquid scintillation counter and is indicative of the extent to which the ligand is bound to the target. However, a SPA requires radio-labeled ligands, which have disposal costs and possible health risks. In addition, a SPA requires the fluoromicrospheres to settle by gravity or be centrifuged, adding an additional step and time to the assay.
Other methods have been developed for detecting kinase activity that are based on luminescence detection, either by bioluminesce or chemiluminescences. Generally, these methods rely on specific substrates and antibodies (Lehel et al. (1977), the use of microchips and fluorescent label probes (Cohen et al. (1999), substrate concentration in a sample (Eu etal al (1999), the use of multiple steps and reagents (Crouch et al. U.S. Pat. No. 6,599,711) or are limited to specific kinases (Sala-Newby et al. (1992).
With phosphorylation events involved in so many cell functions and diseases, identifying transferase activity, especially kinase activity, is tremendously important. Thus, there is a need for enzyme assays that detect protein kinase activity, but that do not require large amounts of costly or highly specialized starting materials, that quickly generate results and are amendable to high through put screening. Additionally, there is a need for assays to rapidly identify activators and inhibitors of kinases. In addition, it is also desirable to provide kits for carrying out such assays. The method, system and kit associated with the present invention may be used for high throughput systems to allow the rapid detection and analysis of effectors, modulators, enhancers and inhibitors of one or more kinase. Moreover, the present invention allows the screening to be completed without the need for specially labeled substrates or antibodies.